ABSTRACT Nearly all of the genetic material among cells within an organism is identical. However, single-nucleotide variants (SNVs), copy-number variants (CNVs), and other structural variants continually accumulate as cells divide during development. This process results in an organism composed of countless cells, each with its own unique genome. Thus, every human is undoubtedly mosaic. Mosaic mutations can go unnoticed, underlie genetic disease or normal human variation, and may be transmitted to the next generation as constitutional variants and cause unexpected intergenerational recurrences of genetic disorders with multiple affected children born to unaffected parents, contrary to Mendelian expectations. The increasing sensitivity and resolution of genomic technologies have identified mosaicism for both SNVs and CNVs. Combined somatic and germline mosaicism has been identified in parents of patients with a number of genetic conditions, thus raising the possibility that mosaic individuals might be detected by routine blood tests rather than requiring direct examination of germ cells. Yet despite its considerable impact on human health, systematic population studies of both germline and somatic mosaicism are lacking. In contrast to recurrent CNVs, usually mediated by nonallelic homologous recombination during meiosis, nonrecurrent CNVs are thought to arise due to DNA replication errors, such as microhomology-mediated break-induced replication during mitosis or non- homologous end joining. Recurrence risks of putatively de novo nonrecurrent CNVs designed to take into account random chance and mosaicism have been previously estimated to be ~ 0.3%. Our preliminary published data on 100 unrelated affected families indicate that somatic mosaicism for CNVs is present in ~ 4% of healthy parents. Currently, the detection rates for CMA and WES are ~ 20% and ~ 25%, respectively, thus in about half of patients no pathogenic variant is found. We propose that some of these patients may have mosaic pathogenic variants that are currently undetected using standard algorithms in CMA and WES. Unrecognized mosaicism could also partially explain variable expressivity and incomplete penetrance. We hypothesize that a significant number of apparently de novo SNVs and CNVs are not meiotic in origin but arise during early post-zygotic mitoses and this can be identified either in the affected patients or their healthy parents. In Aims 1 and 2, we will search for low-level somatic mosaicism in reportedly healthy parents of 500 affected children who have apparent de novo CNVs, indels, or SNVs identified in their affected children. In Aim 2, we will develop a non-invasive prenatal test for detection of recurrent mutations and correlate sperm vs. somatic mosaicism. In Aim 3, we will study somatic mosaic CNVs, indels, and SNVs in patients in whom no nonmosaic pathogenic variant has been found in previous clinical or research genetic testing. We will estimate the currently unrecognized and poorly understood prevalence and clinical consequences of somatic mosaicism and inform about molecular mechanisms of early post-zygotic mutagenesis and human evolution.